The goal of the proposed research is to determine how changes in cis-regulatory sequences alter transcriptional dynamics. Whereas transcription factors are well known to coordinate when a gene is turned on and off, cis-regulatory sequences have the ability to fine-tune how a gene responds to these signals. The precise timing and rate of gene activation/repression are key to a cell's ability to appropriately respond to its biotic and abiotic environment. In microbes, which must survive and compete in ever changing environments, fitness may depend more on gene expression dynamics than expression levels under steady state conditions. To ascertain how changes in cis-regulatory sequences tune gene expression dynamics we will use a high-throughput reporter system capable of testing hundreds of cis-regulatory variants in yeast. We will map causal variants upstream of genes that exhibit allele-specific differences in their gene expression dynamics. In the first aim we will map SNPs and InDels between strains of Saccharomyces cerevisiae that alter expression dynamics following depletion of glucose. In the second aim we will map substitutions between Saccharomyces species responsible for parallel cis-regulatory divergence in their response to temperature. Our central hypothesis is that changes in the relative position of transcription factor binding sites and nucleosomes is the major mode by which gene expression dynamics are altered, whereas changes in the strength of binding sites and nucleosome positioning sequences is the major mode by which gene expression levels are altered. Because studies of cis-regulatory variation have focused on various aspects of expression levels, the completion of the proposed aims on gene expression dynamics will fill an important gap in our understanding of which noncoding variants alter gene expression and how cis-regulatory sequences evolve.